ES2368823T3 - ANTIBODIES OF DEATH RECEIVER 4 (DR4) AND USES OF THE SAME. - Google Patents

Abstract

Death Receptor 4 (DR4) antibodies are provided. The DR4 antibodies may be included in pharmaceutical compositions, articles of manufacture, or kits. Methods of treatment and diagnosis using the DR4 antibodies are also provided.

Description

Death 4 receptor (DR4) antibodies and uses thereof

FIELD OF THE INVENTION

[0001] The present invention relates generally to DR4 antibodies and, in particular, to agonist monoclonal DR4 antibodies.

BACKGROUND OF THE INVENTION

[0002] It is believed that control of the number of cells in mammals is determined, in part, by a balance between cell proliferation and cell death. A form of cell death, sometimes referred to as necrotic cell death, is usually characterized as a pathological form of cell death that results from some trauma or cell injury. On the contrary, there is another "physiological" form of cell death that normally develops in an orderly or controlled manner. This form of ordered or controlled cell death is often referred to as "apoptosis". [see, for example, Barr et al., Bio / Technology, 12: 487-493 (1994); Steller et al., Science, 267: 1445-1449 (1995)]. Apoptotic cell death occurs naturally in many physiological processes, including embryonic development and clonal selection in the immune system [Itoh et al., Cell, 66: 233-243 (1991)]. Reduced levels of apoptotic cell death have been associated with various pathological conditions, including cancer, lupus and herpes virus infection [Thompson, Science, 267: 1456-1462 (1995)]. Increased levels of apoptotic cell death may be associated with other pathological conditions, including AIDS, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, pigmentary retinitis, cerebellar degeneration, aplastic anemia, myocardial infarction, stroke, reperfusion injury and toxin-induced liver disease [see, Thompson, supra].

[0003] Apoptotic cell death is usually accompanied by one or more characteristic morphological and biochemical changes in cells, such as cytoplasm condensation, loss of plasma membrane microvilli, nucleus segmentation, chromosomal DNA degradation or loss of mitochondrial function It is believed that various extrinsic and intrinsic signals trigger or induce such morphological and biochemical cell changes [Raff, Nature, 356: 397-400 (1992); Steller, supra; Sachs et al., Blood, 82: 15 (1993)]. For example, these changes can be triggered by hormonal stimuli, such as glucocorticoid hormones for immature thymocytes, as well as the withdrawal of some growth factors [Watanabe-Fukunaga et al., Nature, 356: 314-317 (1992)]. In addition, it has been described that some identified oncogenes such as myc, rel, and E1A, and tumor suppressors, such as p53, play a role in inducing apoptosis. Similarly, it has been observed that some chemotherapy drugs and some forms of radiation have apoptosis inducing activity [Thompson, supra].

[0004] Various molecules, such as tumor necrosis factor-α ("TNF-α"), tumor necrosis factor-β ("TNF β" or "lymphotoxin-β"), lymphotoxin-β (" LT-β "), CD30 ligand, CD27 ligand, CD40 ligand, OX-40 ligand, 41BB ligand, Apo-1 ligand (also called Fas ligand or CD95 ligand) and Apo-2 ligand (also called TRAIL) have been identified as members of the cytokine family of tumor necrosis factor ("TNF") [See, for example, Gruss and Dower, Blood, 85: 3378-3404 (1995); WO 97/25428 published July 17, 1997; WO 97/01633 published January 16, 1997; Pitti et al., J. Biol. Chem., 271: 12687-12690 (1996); Wiley et al., Immunity, 3: 673-682 (1995); Browning et al., Cell, 72: 847-856 (1993); Armitage et al. Nature, 357: 80-82 (1992)]. Among these molecules, it has been described that TNF-α, TNF-β, ligand CD30, ligand 4-1BB, ligand Apo-1 and ligand Apo-2 (TRAIL) are involved in apoptotic cell death. It has been described that both TNF-α and TNF-β induce apoptotic death in susceptible tumor cells [Schmid et al., Proc. Natl Acad. Sci., 83: 1881 (1986); Dealtry et al., Eur. J. Immunol., 17: 689 (1987)]. Zheng et al. have described that TNF-α is involved in apoptosis after stimulation of CD8 positive T cells [Zheng et al., Nature, 377: 348-351 (1995)]. Other researchers have described that the CD30 ligand may be involved in the removal of self-reactive T cells in the thymus [Amakawa et al., Cold Spring Harbor Laboratory Symposium on Programmed Cell Death, Abstr. No. 10, (1995)].

[0005] Mutations in the mouse Fas / Apo-1 ligand or receptor (called Ipr and gld, respectively) genes have been associated with some autoimmune disorders, indicating that the Apo-1 ligand may play a role in the regulation of the clonal elimination of self-reactive lymphocytes in the periphery [Krammer et al., Curr. Op. Immunol.,

6: 279-289 (1994); Nagata et al., Science, 267: 1449-1456 (1995)]. It has also been described that the Apo-1 ligand induces post-stimulation apoptosis in positive CD4 T cells and B lymphocytes, and may be involved in the removal of activated lymphocytes when their function is no longer necessary [Krammer et al., Supra ; Nagata et al., Supra]. It has been described that mouse agonist monoclonal antibodies that specifically bind to the Apo-1 receptor show cell destruction activity that is comparable or similar to that of TNF-α [Yonehara et al., J. Exp. Med., 169: 1747-1756 (1989)].

[0006] It is believed that the induction of various cellular responses mediated by said cytokines of the TNF family is initiated by their binding to specific cellular receptors. Two distinct TNF receptors of approximately 55 kDa (TNFR1) and 75 kDa (TNFR2) have been identified [Hohman et al., J. Biol. Chem., 264: 14927-14934 (1989); Brockhaus et al., Proc. Natl Acad. Sci., 87: 3127-3131 (1990); EP 417,563, published March 20, 1991] and human and mouse cDNAs corresponding to both types of receptor have been isolated and characterized [Loetscher et al., Cell, 61: 351 (1990); Schall et al., Cell, 61: 361 (1990); Smith et al., Science, 248: 1019-1023 (1990); Lewis et al., Proc. Natl Acad. Sci., 88: 2830-2834 (1991); Goodwin et al., Mol. Cell Biol., 11: 3020-3026 (1991)]. Extensive polymorphisms have been associated with both TNF receptor genes [see, for example, Takao et al., Immunogenetics, 37: 199-203 (1993)]. Both TNFRs share the typical structure of cell surface receptors including extracellular, transmembrane and intracellular regions. The extracellular parts of both receptors are also found naturally as soluble TNF-binding proteins [Nophar, Y. et al., EMBO J., 9: 3269 (1990); and Kohno, T. et al., Proc. Natl Acad. Sci. U.S.A., 87: 8331 (1990)]. More recently, the cloning of recombinant soluble TNF receptors was described by Hale et al. [J. Cell Biochem Supplement 15F, 1991, p. 113 (P424)].

[0007] The extracellular part of type 1 and type 2 TNFR (TNFR1 and TNFR2) contains a repetitive pattern of amino acid sequence of four cysteine-rich domains (CRD) designated from 1 to 4, starting from the NH2 end. Each CRD is approximately 40 amino acids in length and contains 4 to 6 cysteine residues in positions that are well conserved [Schall et al., Supra; Loetscher et al., Supra; Smith et al., Supra; Nophar et al., Supra; Kohno et al., Supra]. In TNFR1, the approximate limits of the four CRDs are as follows: CRD1 amino acids 14 to about 53; CRD2 -amino acids from about 54 to about 97; CRD3-amino acids from about 98 to about 138; CRD4-amino acids from about 139 to about 167. In TNFR2, CRD1 includes amino acids 17 to about 54; CRD2 amino acids from about 55 to about 97; CRD3-amino acids from about 98 to about 140; and CRD4 -amino acids from about 141 to about 179 [Banner et al., Cell, 73: 431-435 (1993)]. The potential role of CRDs in ligand binding has also been described by Banner et al., Supra.

[0008] There is a similar repetitive pattern of CRD in other cell surface proteins, including the p75 nerve growth factor receptor (NGFR) [Johnson et al., Cell, 47: 545 (1986); Radeke et al., Nature, 325: 593 (1987)], the CD40 B cell antigen [Stamenkovic et al., EMBO J., 8: 1403 (1989)], the OX40 T cell antigen [Mallet et al. , EMBO J., 9: 1063 (1990)] and the Fas antigen [Yonehara et al., Supra and Itoh et al., Cell, 66: 233-243 (1991)]. CRD is also found in soluble TFR proteins similar to TNFR (sTNFP) from smallpox viruses Shope and myxoma [Upton et al., Virology, 160: 20-29 (1987); Smith et al., Biochem. Biophys Res. Commun.,

176: 335 (1991); Upton et al., Virology, 184: 370 (1991)]. The optimal alignment of these sequences indicates that the positions of the cysteine residues are well preserved. These receptors are sometimes collectively referred to as members of the TNF / NGF receptor superfamily. Recent studies on p75NGFR showed that the elimination of CRD1 [Welcher, A.A. et al., Proc. Natl Acad. Sci. USA, 88: 159-163 (1991)] or an insertion of 5 amino acids in this domain [Yan, H. and Chao, MV, J. Biol. Chem., 266: 12099-12104 (1991)] had little or no effect on NGF binding [Yan, H. and Chao, MV, supra]. The p75 NGFR contains a proline-rich stretch of approximately 60 amino acids, between its CRD4 and the transmembrane region, which is not involved in NGF binding [Peetre, C. et al., Eur. J. Hematol., 41: 414 -419 (1988); Seckinger, P. et al., J. Biol. Chem., 264: 11966-11973 (1989); Yan, H. and Chao, M.V., supra]. A similar proline-rich region is found in TNFR2 but not in TNFR1.

[0009] Itoh et al. describe that the Apo-1 receptor can signal an apoptotic cell death similar to that indicated by the 55 kDa TNFR1 [Itoh et al., supra]. It has also been described that Apo-1 antigen expression is negatively regulated together with that of TNFR1 when cells are treated with TNF-α or a mouse anti-Apo-1 monoclonal antibody [Krammer et al., Supra; Nagata et al., Supra]. Therefore, some researchers hypothesized that cell lines that co-express Apo-1 and TNFR1 receptors can mediate cell destruction through common signaling pathways [Id.].

[0010] The TNF family ligands identified to date, with the exception of lymphotoxin-α, are transmembrane type II proteins, the C-terminus of which is extracellular. On the contrary, most of the receptors in the TNF receptor family (TNFR) identified to date are transmembrane type I proteins. In the ligand and TNF receptor families, however, the homology identified among the members of the family has been observed mainly in the extracellular domain ("ECD"). Several of the cytokines of the TNF family, including TNF-α, Apo-1 ligand and CD40 ligand, are cleaved proteolytically on the cell surface; The resulting protein in each case usually forms a homotrimeric molecule that functions as a soluble cytokine. The proteins of the TNF receptor family are also normally cleaved proteolytically to release soluble ECDs from the receptor that can function as inhibitors of related cytokines.

[0011] Recently, other members of the TNFR family have been identified. Such newly identified members of the TNFR family include CAR1, HVEM and osteoprotegerin (OPG) [Brojatsch et al., Cell, 87: 845855 (1996); Montgomery et al., Cell, 87: 427-436 (1996); Marsters et al., J. Biol. Chem., 272: 14029-14032 (1997); Simonet et al., Cell, 89: 309-319 (1997)]. Unlike other known TNFR-like molecules, Simonet et al., Supra, describe that OPG does not contain hydrophobic sequences that extend across the membrane.

[0012] In the document Masters et al., Curr. Biol., 6: 750 (1996), the researchers describe a full-length native sequence human polypeptide, called Apo-3, that shows similarity to the TNFR family in their extracellular cysteine-rich repeats and resembles TNFR1 and CD95 in that it contains a cytoplasmic death domain sequence [see also Marsters et al., Curr. Biol., 6: 1669 (1996)]. Other authors also refer to Apo-3 as DR3, wsl-1 and TRAMP [Chinnaiyan et al., Science, 274: 990 (1996); Kitson et al., Nature,

384: 372 (1996); Bodmer et al., Immunity, 6: 79 (1997)].

[0013] Pan et al. have described another member of the TNF receptor family called " DR4 " [Pan et al., Science,

276: 111-113 (1997)]. The DR4 cDNA encodes an open reading frame of 468 amino acids with characteristics characteristic of a cell surface receptor. Pan et al. describe a possible signal peptide present at the beginning of the molecule (amino acids –23 to –1), predicting that the mature protein begins at amino acid 24 (Ala). Residues 108 to 206 contain two pseudo-repetitions rich in cysteine that resemble the corresponding regions in TNFR-1 (four repetitions), DR3 (four repetitions), Fas (three repetitions) and CAR1 (two repetitions). After the transmembrane domain is an intracellular region that contains a 70 amino acid stretch similar to the death domains of TNFR1, DR3, Fas and CAR1. DR4 transcript was detected in the spleen, peripheral blood leukocytes, small intestine and thymus. In addition, DR4 expression was also found in K562 erythroleukemia cells, MCF7 breast carcinoma cells and activated T cells. Pan et al. They also describe that DR4 is believed to be a receptor for the ligand known as Apo-2 or TRAIL ligand.

[0014] In Sheridan et al., Science, 277: 818-821 (1997) and Pan et al., Science, 277: 815-818 (1997), another molecule is described which is believed to be a receptor for the Apo-2 ligand (TRAIL). This molecule is called Apo-2 (as an alternative, it has also been called DR5). Like DR4, it has been described that Apo-2 contains a cytoplasmic death domain and is capable of signaling apoptosis.

[0015] In the document Sheridan et al., Supra, a receptor called DcR1 (or alternatively, Apo2DcR) is described as a potential decoy receptor for the Apo-2 ligand (TRAIL). Sheridan et al. describe that DcR1 can inhibit the function of Apo-2 ligand in vitro. See also, Pan et al., Supra, for a description of the decoy receiver called TRID.

[0016] In the document by Marsters et al., Curr. Biol., 7: 1003-1006 (1997), a receptor called DcR2 is described. Marsters et al. describe that DcR2 contains a cytoplasmic region with a truncated death domain and can act as an inhibitor of Apo-2L inhibitor in vitro.

[0017] For a review of the TNF family of cytokines and their receptors, see Gruss and Dower, supra.

[0018] As currently understood, the cell death program contains at least three important elements - activators, inhibitors and effectors; in C. elegans, these elements are encoded respectively by three genes, Ced-4, Ced-9 and Ced-3 [Steller, Science, 267: 1445 (1995); Chinnaiyan et al., Science, 275: 1122-1126 (1997); Wang et al., Cell, 90: 1-20 (1997)]. Two of the TNFR family members, TNFR1 and Fas / Apo1 (CD95), can activate apoptotic cell death [Chinnaiyan and Dixit, Current Biology, 6: 555562 (1996); Fraser and Evan, Cell; 85: 781-784 (1996)]. It is also known that TNFR1 mediates the activation of the transcription factor, NF-KB [Tartaglia et al., Cell, 74: 845-853 (1993); Hsu et al., Cell, 84: 299-308 (1996)]. In addition to some ECD homology, these two receptors share homology in their intracellular domain (ICD) in an oligomerization interface known as the death domain [Tartaglia et al., Supra; Nagata, Cell, 88: 355 (1997)]. The domains of death are also found in several metazoan proteins that regulate apoptosis, namely, Drosophila, Reaper, and mammalian proteins called FADD / MORT1, TRADD and RIP [Cleaveland and Ihle, Cell, 81: 479- 482 (1995)]. After ligand binding and receptor clustering, TNFR1 and CD95 are believed to recruit FADD in a signaling complex that induces death. CD95 allegedly binds to FADD directly, while TNFR1 binds to FADD indirectly through TRADD [Chinnaiyan et al., Cell, 81: 505-512 (1995); Boldin et al., J. Biol. Chem., 270: 387-391 (1995); Hsu et al., Supra; Chinnaiyan et al., J. Biol. Chem., 271: 4961-4965 (1996)]. It has been described that FADD serves as an adapter protein that recruits the protease related to Ced-3, MACHα / FLICE (caspase 8), in the death signaling complex [Boldin et al., Cell, 85: 803-815 (1996 ); Muzio et al., Cell, 85: 817-827 (1996)]. MACHα / FLICE appears to be the trigger that initiates a cascade of apoptotic proteases, including the interleukin-1β conversion enzyme (ICE) and CPP32 / Yama, which can execute some critical aspects of the cell death program [Fraser and Evan, supra] .

[0019] Recently it was described that programmed cell death involves the activity of members of a family of cysteine proteases related to the C. elegans cell death gene, ced-3, and the IL-1 conversion enzyme of mammal, ICE. The activity of the ICE and CPP32 / Yama proteases can be inhibited by the product of the cowpox virus gene, crmA [Ray et al., Cell, 69: 597-604 (1992); Tewari et al., Cell, 81: 801-809 (1995)]. Recent studies show that CrmA can inhibit cell death induced by TNFR1 and CD95 [Enari et al., Nature, 375: 78-81 (1995); Tewari et al., J. Biol. Chem., 270: 3255-3260 (1995)].

[0020] As recently reviewed by Tewari et al., TNFR1, TNFR2 and CD40 modulate the expression of proinflammatory and costimulatory cytokines, cytokine receptors and cell adhesion molecules through the activation of the transcription factor, NF-kB (Tewari et al., Curr. Op. Genet. Develop., 6: 39-44 (1996)]. NF-kB is the prototype of a family of dimeric transcription factors whose subunits contain conserved Rel regions [Verma et al. , Genes Develop., 9: 2723-2735 (1996); Baldwin, Ann. Rev. Immunol., 14: 649-681 (1996).] In its latent form, NF-kB complexes with members of the inhibitor family of IkB; after inactivation of IκB in response to certain stimuli, the released NF-κB translocates to the nucleus where it binds to specific DNA sequences and activates gene transcription. WO98 / 32856, citable under Art 54 ( 3) EPC refers to the DR4 polypeptide.

SUMMARY DESCRIPTION OF THE INVENTION

[0021] The present invention relates to an isolated agonist monoclonal antibody that (a) specifically binds to a DR4 polypeptide comprising amino acid residues 24-218, 1-218, or 1-468 of Fig. 1 (SEQ ID NO: 1),

(b) induces apoptosis in at least one type of mammalian cell that expresses the DR4 polypeptide and (c) does not bind DcR1 or DcR2, and thus are useful in the treatment of various diseases and pathological conditions, including Cancer.

[0022] The present invention also provides hybridoma cell lines that produce DR4 monoclonal antibodies.

[0023] The present invention also provides compositions comprising one or more DR4 antibodies and a carrier, such as a pharmaceutically acceptable carrier. In one embodiment, said composition may be included in a manufacturing article or kit.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024]

Figure 1 shows the nucleotide sequence (SEQ ID No. 2) of a cDNA for human DR4 and its derived amino acid sequence (SEQ ID No. 1). The respective nucleotide and amino acid sequences for human DR4 are also described in Pan et al., Science, 276: 111 (1997).

Figure 2 shows the FACS analysis of two anti-DR4 antibodies, 4E7.24.3 and 4H6.17.8 (illustrated by bold lines) compared to IgG controls (expressed in dots) in human 9D cells.

Figure 3 is a graph showing the percentage (%) of apoptosis induced in 9D cells by DR4, 4E7.24.3 and 4H6.17.8 antibodies, in the absence of goat anti-mouse IgG Fc.

Figure 4 is a bar chart showing the percentage (%) of apoptosis, compared to Apo-2L in 9D cells by DR4, 4E7.24.3 and 4H6.17.8 antibodies, in the presence or absence of anti-IgG Fc goat mouse

Figure 5 is a bar chart showing the ability of DR4 antibody 4H6.17.8 to block Apo-2L-induced apoptosis in 9D cells.

Figure 6 is a graph showing the results of an ELISA that analyzes the binding of DR4, 4E7.24.3 and 4H6.17.8 antibodies, to DR4 and other known Apo-2L receptors, referred to as Apo-2, DcR1, and DcR2.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

I. Definitions

[0025] As used in the present invention, the term "Apo-2 ligand" or "Apo-2L" (also known as TRAIL) refers to a specific member of the family of tumor necrosis factor (TNF) ligands that induces apoptosis in a set of cell lineages [see WO 97/25428 published July 17, 1997; Pitti et al., J. Biol. Chem, 271: 12687 (1996); Marsters et al., Curr. Biol., 6: 79 (1997); Wiley, S. et al., Immunity, 3: 637 (1995)].

[0026] A receptor for Apo-2L has been identified and has been called DR4, a member of the TNF receptor family that contains a cytoplasmic "death domain" capable of attracting the cell's suicide apparatus [see Pan et al., Science, 276: 111 (1997)]. The term "Death Receiver 4" or "DR2", when used in the present invention, comprises native sequence DR4 and DR4 variants (defined hereinafter). These terms comprise DR4 expressed in a variety of mammals, including humans. DR4 can be expressed endogenously as it appears naturally in a variety of human tissue lineages or it can be expressed by recombinant or synthetic methods. A "native sequence DR4" comprises a polypeptide having the same amino acid sequence as a nature-derived DR4. Thus, a native sequence DR4 can have the natural DR4 amino acid sequence of any mammal. Said native sequence DR4 can be isolated from nature or can be produced by recombinant or synthetic means. The term "native sequence DR4" specifically comprises natural truncated or secreted forms of DR4 (for example, a soluble form containing, for example, an extracellular domain sequence), natural variant forms (eg alternative splicing forms ) and natural allelic variants of DR4. In one embodiment of the invention, the native sequence DR4 is a mature or full length native sequence DR4 comprising amino acids 1 to 468 of Figure 1 (SEQ ID No. 1)

[0027] The terms "extracellular domain" or "ECD" refers in the present invention to a form of DR4 that is essentially free of the transmembrane and cytoplasmic domains of DR4. Typically, the DR4 ECD will have less than 1% of said transmembrane and / or cytoplasmic domains and, preferably, will have less than 0.5% of said domains. Optionally, the DR4 ECD will comprise amino acid residues 1 to 218 or residues 24 to 218 of Figure 1 (SEQ ID NO: 1).

[0028] "DR4 variant" means a biologically active DR4 that has at least about 80% or 85% identity in the amino acid sequence with DR4 having the deduced amino acid sequence shown in Figure 1 (SEC ID No: 1) for a full length native human sequence DR4. Such variants of DR4 include, for example, DR4 polypeptides in which one or more amino acid residues are added, or removed at the Non-C-terminal end of the sequence of Figure 1 (SEQ ID NO: 1) . Normally, a variant of DR4 will have at least about 80% identity in the amino acid sequence, more preferably at least about 90% identity in the amino acid sequence, and even more preferably at least about 95%. of identity in the amino acid sequence with the amino acid sequence of Figure 1 (SEQ ID No: 1).

[0029] The "percentage (%) of identity in the amino acid sequence" with respect to the DR4 sequences identified in the present invention is defined as the percentage of amino acid residues in a candidate sequence that are identical with amino acid residues. in the sequence of DR4, after the alignment of the sequences and the introduction of spaces, if necessary, to achieve the maximum percentage of identity in the sequence, and without considering any conservative substitution as part of the identity of the sequence. Alignment with the objective of determining the percentage of identity in the amino acid sequence can be achieved in several ways that are within the art, for example, using publicly available computer software, such as ALIGNTM or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm necessary to achieve maximum alignment over the full length of the sequences being compared.

[0030] " Isolated " when used to describe the various polypeptides described in the present invention, it means a polypeptide that has been identified and separated and / or recovered from a component of its natural environment. Contaminating components of their natural environment are materials that would normally interfere with the diagnostic or therapeutic uses of the polypeptide, and may include enzymes, hormones and other protein or non-protein solutes. In preferred embodiments, the polypeptide will be purified (1) to a degree sufficient to obtain at least 15 residues of an internal amino acid sequence or of the N-terminus using a rotating cup sequencer, or (2) to homogeneity by SDS- PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver staining. An isolated polypeptide includes a polypeptide in situ within recombinant cells, since at least one component of the natural environment of DR4 will be missing. However, normally, the isolated polypeptide will be prepared by at least one purification step.

[0031] The terms "agonist" and "agonistic" when used in the present invention refer to or describe a molecule that is capable, directly or indirectly, of inducing, causing or substantially increasing the biological activity or activation of DR4.

[0032] The terms "antagonist" and "antagonist" when used in the present invention refer to or describe a molecule that is capable, directly or indirectly, of counteracting, substantially reducing or substantially inhibiting the biological activity or activation of DR4.

[0033] The term "antibody" is used in the broadest sense and specifically covers individual anti-DR4 monoclonal antibodies (including agonist, antagonist, and neutralizing and blocking antibodies) and anti-DR4 antibody compositions with polyepitopic specificity. "Antibody", as used in the present invention, includes intact immunoglobulin molecules or antibody polyclonal antibodies, multispecific antibodies (ie, bispecific antibodies formed from at least two intact antibodies) and immunoglobulin fragments (such as Fab , F (ab ') 2, or Fv), provided they show any of the desired agonistic properties described in the present invention.

[0034] Antibodies are usually proteins or polypeptides that show specificity of binding to a specific antigen. Native antibodies are usually heterotetrameric glycoproteins, composed of two identical light chains (L) and two identical heavy chains (H). Typically, each light chain is linked to a heavy chain by a covalent disulfide bond, while the number of disulfide bonds varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intracatenary disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a set of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain aligns with the first constant domain of the heavy chain, and the variable domain of the light chain aligns with the variable domain of the heavy chain. It is believed that there are specific amino acid residues that form an interface between the light chain and heavy chain variable domains [Chothia et al., J. Mol. Biol., 186: 651-663 (1985); Novotny and Haber, Proc. Natl Acad. Sci. USA, 82: 4592-4596 (1985)]. The light chains of antibodies of any vertebrate species can be assigned to one of two clearly distinct types, called kappa (6) and lambda (8), based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these can be further divided into subclasses (isotypes), for example, IgG-1, IgG-2, IgG-3, and IgG-4 ; IgA-1 and IgA-2. The constant heavy chain domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.

[0035] "Antibody fragments" they comprise a part of an intact antibody, generally the antigen-binding region or variable of the intact antibody. Examples of antibody fragments include Fab, Fab ’, F (ab’) 2, and Fv, diabodies, single chain antibody molecules and multispecific antibodies formed from antibody fragments.

[0036] The term "variable" is used in the present invention to describe certain parts of the variable domains that differ in the sequence between the antibodies and are used in the binding and specificity of each particular antibody by its particular antigen. However, the variability is not normally distributed uniformly throughout the variable domains of the antibodies. It is usually concentrated in three segments called complementarity determining regions (CDR) or hypervariable regions in the variable domains of both the light chain and the heavy chain. The most highly conserved parts of the variable domains are called the framework (“framework”) (FR). The variable domains of native heavy and light chains each comprise four FR regions, which mostly adopt a β-sheet configuration, connected by three CDRs, which form loops that connect, and in some cases are part of, the β-sheet structure. The CDRs in each chain are held together in proximity by the FR regions and, with the CDRs of the other chain, contribute to the formation of the antigen binding site of the antibodies [see, Kabat,

E.A. et al., Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, MD (1987)]. Constant domains are not directly involved in the binding of an antibody to an antigen, but show several effector functions, such as the participation of the antibody in antibody-dependent cellular toxicity.

[0037] The term "monoclonal antibody", as used in the present invention, refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, each of the antibodies comprising the population are identical except for possible natural mutations that may be present in smaller amounts. Monoclonal antibodies are very specific, targeting a single antigenic site. In addition, unlike conventional (polyclonal) antibody preparations, which usually include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant in the antigen.

[0038] The monoclonal antibodies of the present invention include chimeric, hybrid and recombinant antibodies produced by splicing a variable domain (including hypervariable) of an anti-DR4 antibody with a constant domain (eg, "humanized antibodies"), or a light chain with a heavy chain, or a chain of a species with a chain of another species, or fusions with heterologous proteins, regardless of the species of origin or the designation of the immunoglobulin class or subclass, as well as fragments of antibody (eg, Fab, F (ab ') 2 and Fv), provided they show the desired biological activity. See, for example, US Patent No. 4,816,567 and Mage et al., In Monoclonal Antibody Production Techniques and Applications. pages. 79-97 (Marcel Dekker, Inc .: New York, 1987).

[0039] Thus, the "monoclonal" modifier indicates the character of the antibody to be obtained from a substantially homogeneous population of antibodies and it should not be construed that the production of antibodies is required by any particular procedure. For example, the monoclonal antibodies to be used according to the present invention can be manufactured by the hybridoma method described for the first time by Kohler and Milstein, Nature, 256: 495 (1975), or they can be manufactured by recombinant DNA methods such as those described in U.S. Patent 4,816,567. The "monoclonal antibodies" they can also be isolated from phage libraries generated using, for example, the techniques described in McCafferty et al., Nature, 348: 552554 (1990).

[0040] The "humanized" forms of non-human antibodies (e.g. murine) are specific chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab ', F (ab') 2 or other sub-sequences of antigen-binding antibodies) that contain a minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (receptor antibody) in which residues of a receptor complementarity determining region (CDR) are replaced by residues of a CDR of a non-human species (donor antibody) such as a mouse , a rat or a rabbit that has the desired specificity, affinity and capacity. In some cases, residues in the framework region (FR framework) of Fv of the human immunoglobulin are replaced by the corresponding non-human residues. In addition, the humanized antibody may comprise residues that are neither found in the recipient antibody nor in the imported CDR sequences or in the framework sequences. These modifications are made to refine and optimize antibody performance. In general, the humanized antibody will comprise substantially all, of at least one variable domain, and usually two, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a consensus sequence of human immunoglobulin. The humanized antibody will also optimally comprise at least a part of an immunoglobulin constant region (Fc), usually that of a human immunoglobulin.

[0041] "Biologically active" and "desired biological activity" for the purposes of the present invention means that it has the ability to modulate apoptosis (either agonistically or stimulantly or in antagonistic or blocking form) in vivo or ex vivo in at least one type of mammalian cell

[0042] The terms "apoptosis" and "apoptotic activity" they are used in a broad sense and refer to the orderly or controlled form of cell death in mammals that are usually accompanied by one or more characteristic cellular changes, including cytoplasm condensation, loss of plasma membrane microvilli , the segmentation of the nucleus, the degradation of chromosomal DNA or loss of mitochondrial function. This activity can be determined and measured, for example, by cell viability analysis, FACS analysis or DNA electrophoresis, all known in the art.

[0043] The terms "treat," "treatment" and "therapy", as used in the present invention, refers to curative therapy, prophylactic therapy and preventive therapy.

[0044] The term " mammal " As used in the present invention, it refers to any mammal classified as a mammal, including humans, cows, horses, dogs and cats. In a preferred embodiment of the invention, the mammal is a human.

II.

Compositions and methods of the invention

TO.

DR4 antibodies

[0045] The present invention relates to agonist monoclonal antibodies that (a) specifically bind to a DR4 polypeptide comprising amino acid residues 24-218, 1-218, or 1-468 of Figure 1 (SEQ ID NO: one),

(b) induce apoptosis in at least one type of mammalian cell that expresses the DR4 polypeptide, and (c) do not bind DcR1 or DcR2. For reference, other types of antibody are described below.

one.

Polyclonal antibodies

[0046] The methods of preparing polyclonal antibodies are known to the person skilled in the art. Polyclonal antibodies can be developed in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Usually, the immunizing agent and / or adjuvant will be injected into the mammal by multiple subcutaneous or intraperitoneal injections. The immunizing agent may include the DR4 polypeptide (or an DR4 ECD) or a fusion protein thereof. It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal that is immunized. Examples of such immunogenic proteins include, but are not limited to, limpet hemocyanin, serum albumin, bovine thyroglobulin, and soy trypsin inhibitor. Examples of adjuvants that can be used include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl lipid A, synthetic trehalose dichlorinomycolate). The immunization protocol can be selected by a person skilled in the art without extensive experimentation. Next, blood can be drawn to the mammal and the serum analyzed for antibody titration. If desired, the mammal can be stimulated again until the antibody titre increases or until it reaches a constant level ("plateaus").

2.

Monoclonal antibodies

[0047] The antibodies of the present invention are monoclonal antibodies.

[0048] Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, supra. In a hybridoma procedure, a mouse, hamster or other appropriate host animal is usually immunized with an immunizing agent to obtain lymphocytes that produce

or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

[0049] The immunizing agent will usually include the DR4 polypeptide (or an DR4 ECD) or a fusion protein thereof, such as a DR4-IgG ECD fusion protein. The immunizing agent may alternatively comprise a fragment or part of DR4 having one or more amino acids that participate in the binding of Apo-2L to DR4. In a preferred embodiment, the immunizing agent comprises a sequence of the DR4 extracellular domain fused to an IgG sequence, as described in Example 1.

[0050] Generally, peripheral blood lymphocytes ("PLBs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if sources of non-human mammals are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusion agent, such as polyethylene glycol, to form a hybridoma cell [Goding, Monoclonal Antibodies: Principles and Practice, Academia Press, (1986), pages 59-103 ]. Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Typically, rat or mouse myeloma cell lines are used. Hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of non-fused immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for hybridomas will usually include hypoxanthine, aminopterin and thymidine ("HAT medium"), whose substances prevent the growth of deficient cells in HGPRT.

[0051] Preferred immortalized cell lines are those that efficiently fuse, support a stable high level of antibody expression by the selected antibody producing cells, and are sensitive to a medium such as HAT medium. The most preferred immortalized cell lines are murine myeloma lines, which can be obtained, for example, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia. An example of said murine myeloma cell line is P3X63AgU.1 described in example 2 below. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies [Kozbor, J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pages, 51-63].

[0052] Next, in the culture medium in which the hybridoma cells are cultured, the presence of monoclonal antibodies directed against DR4 can be analyzed. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme linked immunosorbent assay (ELISA). Such techniques and tests are known in the state of the art. The binding affinity of the monoclonal antibody can be determined, for example, by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107: 220 (1980).

[0053] After identifying the desired hybridoma cells, the clones can be subcloned by limiting dilution procedures and can be developed by standard procedures [Goding, supra]. Suitable culture media for this purpose include, for example, Eagle medium modified by Dulbecco and RPMI-1640 medium. Alternatively, hybridoma cells can be developed in vivo as ascites fluid in a mammal.

[0054] Monoclonal antibodies secreted by subclones can be isolated or purified from the culture medium

or ascites fluid by conventional immunoglobulin purification procedures, such as, for example, A-sepharose protein, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0055] Monoclonal antibodies can also be manufactured by recombinant DNA methods, such as those described in US Patent No. 4,816,567. The DNA encoding the monoclonal antibodies of the present invention can be easily isolated and sequenced using conventional methods (for example, by the use of oligonucleotide probes that are capable of specifically binding to genes encoding the light and heavy chains of murine antibodies) . Hybridoma cells serve as a preferred source of said DNA. Once isolated, the DNA can be placed in expression vectors, which are then transfected into host cells, such as simian COS cells, Chinese hamster ovary (CHO) cells or myeloma cells that do not produce protein in any way. of immunoglobulin, to obtain the synthesis of monoclonal antibodies in recombinant host cells. DNA can also be modified, for example, by replacing the coding sequence for human heavy and light chain constant domains instead of murine homologous sequences [US Patent No. 4,816,567; Morrison et al., Supra] or covalently binding to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Said non-immunoglobulin polypeptide may be substituted for the constant domains of an antibody of the present invention, or it may be substituted for the variable domains of an antigen combination site of an antibody of the invention to create a bivalent chimeric antibody.

[0056] As described in the following examples, several anti-DR4 monoclonal antibodies have been identified and prepared. Some of these antibodies, referred to herein as 4E7.24.3 and 4H6.17.8, have been deposited with the ATCC and assigned no. of tank access HB-12454 and HB-12455, respectively. In one embodiment, the monoclonal antibodies of the present invention will have the same biological characteristics as the monoclonal antibodies secreted by the hybridoma cell line or lines deposited under the no. Access HB-12454 or HB-12455. The term "biological characteristics" is used to refer to the in vitro and / or in vivo activities or properties of the monoclonal antibody, such as the ability to specifically bind DR4 or to block, induce or enhance the activation of DR4 (or related activities of DR4). As described herein, the monoclonal antibody 4E7.24.3 is characterized by specifically binding to DR4 (and having no specificity for binding by Apo-2, DcR1 or DcR2), capable of inducing apoptosis, and not being able of blocking DR4. Monoclonal antibody 4H6.17.8 is characterized by specifically binding to DR4 (and not having some cross-reactivity with Apo-2, but not with DcR1 or DcR2), capable of inducing apoptosis and capable of blocking DR4. Optionally, the monoclonal antibodies of the present invention will bind to the same epitope or epitopes as the 4E7.24.3 or 4H6.17.8 antibodies described in the present invention. This can be determined by performing various tests, such as those described in the present invention and in the Examples. For example, to determine whether a monoclonal antibody has the same specificity as the 4E7.24.3 or 4H6.17.8 antibodies specifically described, its activity can be compared in the DR4 blockade assays and the induction of apoptosis, such as those described in The following examples.

[0057] The antibodies of the present invention may also comprise monovalent antibodies. The procedures for preparing monovalent antibodies are known in the state of the art. For example, one method involves recombinant expression of the light chain and the modified heavy chain of the immunoglobulin. The heavy chain is generally truncated at any point in the Fc region to avoid crosslinking of the heavy chain. Alternatively, the relevant cysteine residues are replaced by another amino acid residue or removed to avoid crosslinking.

[0058] In vitro procedures are also suitable for preparing monovalent antibodies. The digestion of antibodies to produce fragments thereof, particularly Fab fragments, can be performed using routine techniques known in the state of the art. For example, digestion can be done with papain. Some examples of papain digestion are described in WO 94/29348 published 12/22/94 and US Patent No. 4,342,566. Papain digestion of antibodies usually produces two identical antigen-binding fragments, called Fab fragments, each with a single antigen-binding site, and a residual Fc fragment. Pepsin treatment produces an F (ab ’) 2 fragment that has two antigen-combining sites and is still capable of forming cross-links with the antigen.

[0059] Fab fragments produced in the digestion of the antibody also contain the constant domains of the light chain and the first constant domain (CH1) of the heavy chain. Fab ’fragments differ from Fab fragments in the addition of a few residues at the carboxylic end of the CH1 domain of the heavy chain including one or more cysteines of the hinge region of the antibody. Fab’-SH is the designation in the present invention for a Fab ’in which the cysteine residue or residues of the constant domains carry a free thiol group. F (ab ’) 2 fragments of the antibodies were originally produced as pairs of Fab’ fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0060] Single chain Fv fragments can also be produced, as described in Iliades et al., FEBS Letters, 409: 437-441 (1997). The coupling of said single chain fragments using several linkers is described in Kortt et al., Protein Engineering, 10: 423-433 (1997).

[0061] In addition to the antibodies described above, it is contemplated that chimeric or hybrid antibodies can be prepared in vitro using methods known in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by the formation of a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate.

3. Humanized antibodies

[0062] The DR4 antibodies of the present invention may further comprise humanized antibodies or human antibodies. Humanized forms of non-human antibodies (eg, murine) are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab ', F (ab') 2 or other antigen binding sub-sequences of the antibodies) that contain a minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (receptor antibody) in which residues of a receptor complementarity determining region (CDR) are replaced by residues of a CDR of a non-human species (donor antibody), such as mouse, rat

or rabbit that have the specificity, affinity and capacity desired. In some cases, the residues of the Fv framework of the human immunoglobulin are replaced by the corresponding non-human residues. Humanized antibodies may also comprise residues that are neither found in the recipient antibody nor in the sequences imported from the CDR or framework. In general, the humanized antibody will comprise substantially all of at least one, and usually two, variable domains, in which all or substantially all CDR regions correspond to those of a non-human immunoglobulin and all or substantially all regions of FR are those of a consensus sequence of human immunoglobulin. The optimally humanized antibody will also comprise at least a portion of a constant region of immunoglobulin (Fc), usually that of a human immunoglobulin [Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2: 593-596 (1992)].

[0063] Methods for humanizing non-human antibodies are known in the state of the art. Generally, a humanized antibody has one or more amino acid residues introduced therein from an origin that is non-human. These non-human amino acid residues are often referred to as "imported" residues, which are usually acquired from a "imported" variable domain. Humanization can be performed essentially following the procedure of Winter et al. [Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, said "humanized" antibodies are chimeric antibodies (US Patent No. 4,816,567), in which substantially less than one intact human variable domain has been replaced by the corresponding sequence of a non-human species. In practice, humanized antibodies are usually human antibodies in which some CDR residues and possibly some FR residues are replaced by residues of similar sites in rodent antibodies.

[0064] The choice of human variable domains, both light and heavy, for use in the manufacture of humanized antibodies is very important to reduce antigenicity. According to the "optimal fit" procedure, the variable domain sequence of a rodent antibody is screened against a whole library of known human variable domain sequences. Next, the human sequence closest to that of a rodent is accepted as the human framework region (FR) for the humanized antibody [Sims et al., J. Immunol.,

151: 2,296 (1993); Chothia and Lesk, J. Mol. Biol., 196: 901 (1987)]. Another method uses a particular framework region derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework region can be used for several different humanized antibodies [Carter et al., Proc. Natl Acad. Sci. USA, 89: 4,285 (1992); Presta et al., J. Immunol., 151: 2,623 (1993)].

[0065] It is also important that antibodies be humanized while maintaining high affinity for the antigen and other favorable biological properties. To achieve this objective, according to a preferred method, humanized antibodies are prepared by a process of analysis of the parental sequences and of several humanized conceptual products using three-dimensional models of the parental and humanized sequences. Three-dimensional models of immunoglobulin are normally available and are known to those skilled in the art. There is an availability of computer programs that illustrate and show probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. The exploration of these structures allows analyzing the probable role of the residues in the functioning of the candidate immunoglobulin sequence, that is, the analysis of residues that influence the ability of the candidate immunoglobulin to bind to its antigen. Thus, FR residues can be selected and combined from the consensus sequence and the import sequence, so that the desired characteristic of the antibody is achieved, such as an increase in affinity for the target antigen or antigens. In general, CDR residues are directly and more substantially involved in the influence on antigen binding [see, WO 94/04679 published March 3, 1994].

[0066] Transgenic animals (eg, mice) that are capable, after immunization, of producing a complete repertoire of human antibodies in the absence of endogenous immunoglobulin production can be used. For example, it has been described that homozygous elimination of the binding region (JH) gene from the antibody heavy chain in germline mutant chimeric mice results in a complete inhibition of endogenous antibody production. The transfer of the human germline immunoglobulin gene series in said germline mutant mice will result in the production of human antibodies after antigen stimulation [see, for example, Jakobovits et al., Proc. Natl, Acad. Sci. USA,

90: 2,551-255 (1993); Jakobovits et al., Nature, 362: 255-258 (1993); Bruggermann et al., Year in Immuno., 7: 33 (1993)]. Human antibodies can also be produced in phage expression libraries [Hoogenboom and Winter, J. Mol. Biol., 227: 381 (1992); Marks et al., J. Mol. Biol., 222: 581 (1991)]. The techniques of Cote et al. and Boerner et al. They are also available for the preparation of human monoclonal antibodies (Cote et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147 (1): 86-95 (1991)].

4. Bispecific Antibodies

[0067] Bispecific antibodies are monoclonal antibodies, preferably human or humanized, which have binding specificity for at least two different antigens. In the present case, one of the binding specificities is for DR4, the other is for any other antigen, and preferably for a cell or receptor surface protein or a receptor subunit.

[0068] Methods for making bispecific antibodies are known in the state of the art. Typically, the recombinant production of bispecific antibodies is based on the coexpression of two pairs of light chains / heavy chains of the immunoglobulin, where the two heavy chains have different specificities [Millstein and Neck, Nature, 305: 537-539 (1983)] . Because of the random distribution of the heavy and light chains of the immunoglobulin, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule is normally carried out by affinity chromatography steps. Similar procedures are described in WO 93/08829, published May 13, 1993, and in Traunecker et al., EMBO J., 10: 3,655-3,659 (1991).

[0069] The variable domains of the antibody with the desired binding specificities (antibody-antigen combination sites) can be fused to sequences from the constant domains of the immunoglobulin. Fusion takes place, preferably, with a constant domain of the immunoglobulin heavy chain, comprising at least part of the hinge, CH2, and the CH3 regions. It is preferred that it has the first constant region of the heavy chain (CH1) that contains the site necessary to join the light chain present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and co-transfected into a suitable host organism. For more details on how to generate bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121: 210 (1986).

5.

Heteroconjugate Antibodies

[0070] Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently bound antibodies. Such antibodies have been proposed, for example, to direct cells of the immune system towards unwanted cells [US Patent 4,676,980] and for the treatment of HIV infection [WO 91/00360; WO 92/200373; EP 03089]. It is contemplated that antibodies can be prepared in vitro using methods known in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those described, for example, in US Patent 4,676,980.

6.

Triabodies

[0071] "Triabodies" are also within the scope of the present invention. Such antibodies are described for example in Iliades et al., Supra and Korrt et al., Supra.

B. Uses for DR4 antibodies

[0072] The DR4 antibodies of the present invention have several utilities. For example, agonistic DR4 antibodies can be used in methods for the treatment of pathological conditions, such as malignant tumors. The diagnosis of these conditions is in the usual capacity of a doctor or clinician. In the methods, the agonistic DR4 antibody is administered to a mammal, alone or in combination with other therapeutic agents or techniques.

[0073] The antibody is preferably administered to the mammal in a carrier; preferably a pharmaceutically acceptable carrier. Suitable carriers and their formulations are described in Remington’s Pharmaceutical Sciences, 16th ed., 1980, Mack Publishing Co., edited by Oslo et al. Usually, an appropriate amount of a pharmaceutically acceptable salt is used in the formulation so that the formulation is isotonic. Examples of carriers include saline, Ringer's solution and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. Some additional carriers include prolonged release preparations, such as semipermeable matrices of solid hydrophobic polymers containing the agonist, whose matrices are in the form of shaped articles, for example, films, liposomes or microparticles. It will be apparent to those skilled in the art that certain carriers may be more preferable depending on, for example, the route of administration and the concentration of antibody that is administered.

[0074] The antibody can be administered to the mammal by injection (for example, intravenous, intraperitoneal, subcutaneous, intramuscular, intraportal), or by other procedures, such as infusion, which ensure its release into the bloodstream efficiently. The antibody can also be administered by isolated perfusion techniques, such as perfusion of isolated tissue, to exert local therapeutic effects. Local or intravenous injection is preferred.

[0075] Dosages and effective guidelines for antibody administration can be empirically determined, and the performance of such determinations is part of the skill of the person skilled in the art. Those skilled in the art will understand that the dosage of antibody to be administered will vary depending, for example, on the mammal that will receive the antibody, the route of administration, the particular type of antibody used and other drugs that are administered to the mammal. Guidelines for selecting appropriate doses for antibodies are found in the literature on the therapeutic uses of antibodies, for example, Handbook of Monoclonal Antibodies, Ferrone et al., Eds., Noges Publications, Park Ridge, NJ, (1985) , chapter 22 and pages 303-357; Smith et al, Antibodies in Human Diagnosis and Therapy, Haber et al., Eds., Raven Press, New York (1977), pages 365-389. A usual daily dosage of the antibody used could only vary between about 1 µg / kg to 100 mg / kg body weight or more per day, depending on the factors mentioned above.

[0076] The antibody can also be administered to the mammal in combination with effective amounts of one or more other therapeutic agents. Among the one or more other therapeutic agents or therapies may be included, but not limited to, chemotherapy, radiotherapy, immunoadjuvants and cytokines. Other agents known to induce apoptosis in mammalian cells can also be used and such agents include TNF-alpha, TNF-beta, CD30 ligand, 4-1BB ligand and Apo-2 ligand.

[0077] Chemotherapies contemplated by the present invention include chemical substances or drugs that are known in the state of the art and are commercially available, such as doxorubicin, 5-fluorouracil, etoposide, camptothecin, Leucovorin, cytosine arabinoside , cyclophosphamide, thiotepa, busulfan, cytoxin, taxol, methotrexate, cisplatin, melphalan, vinblastine and carboplatin. The preparation and dosage guidelines for such chemotherapy can be used according to the manufacturer's instructions or determined empirically by the person skilled in the art. The preparation and dosage guidelines for such chemotherapy are also described in Chemotherapy Service Ed., M.C. Perry, Williams & Wilkins, Baltimore, MD (1992).

[0078] Chemotherapy is preferably administered in a pharmaceutically acceptable carrier, such as those described above. The mode of administration of chemotherapy may be the same as that used for the DR4 antibody or it may be administered to the mammal through a different mode. For example, the DR4 antibody can be injected while chemotherapy is administered orally to the mammal.

[0079] Radiation therapy can be administered to the mammal according to the protocols commonly used in the art and known to the person skilled in the art. Such therapy may include radiation with cesium, iridium, iodine or cobalt. Radiation therapy can be radiation throughout the body, or it can be directed locally to a specific site or tissue in or on the body. Usually, radiation therapy is given in pulses for a period of about 1 to about 2 weeks. Radiation therapy can be administered, however, for longer periods of time. Optionally, radiotherapy can be administered as a single dose or as multiple sequential doses.

[0080] The antibody can be administered sequentially or simultaneously with the other or more therapeutic agents. The amounts of antibody and therapeutic agent depend, for example, on the type of drug used, the pathological condition being treated and the guidelines and routes of administration, but, in general, would be lower than if they were used individually.

[0081] After administration of the antibody to the mammal, the physiological condition of the mammal can be monitored in several ways known to the person skilled in the art.

[0082] It is contemplated that DR4 antibody blocking can also be used in therapy. For example, a blocking DR4 antibody can be administered to a mammal (as described above) to block receptor binding to Apo-2L, thereby increasing the bioavailability of Apo-2L to induce apoptosis.

[0083] In another embodiment, methods for using the antibody in diagnostic assays are provided. For example, antibodies can also be used in diagnostic assays for the detection of DR4 overexpression in specific cells and tissues. Various diagnostic test techniques known in the state of the art can be used, such as in vivo imaging tests, competitive binding assays, "sandwich" tests. direct or indirect and immunoprecipitation tests performed in heterogeneous phases or in homogeneous phases [Zola, Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc. (1987) pages 147-158]. The agonists used in diagnostic tests may be marked with a detectable part. The detectable part should be able to produce, directly or indirectly, a detectable signal. For example, the detectable part may be a radioisotope, such as 3H, 14C, 32P, 35S, or 125I, a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase. Any method known in the state of the art can be used to conjugate the antibody with the detectable part, including those procedures described by Hunter et al., Nature, 144: 945 (1962); David et al., Biochemistry, 13: 1,014 (1974); Pain et al., J. Immunol, Meth., 40: 219 (1981); and Nygren, J. Histochem. and Cytochem., 30: 407 (1982).

[0084] DR4 antibodies are also useful for affinity purification of DR4 from a recombinant or naturally occurring cell culture. In this process, antibodies against DR4 are immobilized on a suitable support, such as a Sephadex resin or a filter paper, using procedures well known in the state of the art. Next, the immobilized antibody is contacted with a sample containing the DR4 to be purified, and then the support is cleaned with a suitable solvent that will substantially remove all of the sample material except the DR4 that is bound to the immobilized antibody. Finally, the support is washed with another suitable solvent that will release DR4 from the antibody.

[0085] In a further embodiment, articles of manufacture and kits containing materials useful for the treatment of pathological conditions or the detection or purification of DR4 are provided. The article of manufacture comprises a container with a label. Suitable containers include, for example, bottles, vials and test tubes. The containers may be formed of a series of materials, such as glass or plastic. The container contains a composition that includes an active agent that is effective for the treatment of pathological conditions or the detection or purification of DR4. The active agent in the composition is a DR4 antibody and preferably, comprises monoclonal antibodies to DR4. The label on the container indicates that the composition is used for the treatment of pathological conditions or the detection or purification of DR4, and may also indicate guidelines for in vivo or in vitro use, as described above.

[0086] The kit will comprise the container described above and a second container comprising a buffer. Other desirable materials from a commercial and user point of view may also be included, including other buffers, diluents, filters, needles, syringes and package leaflets with the instructions for use.

[0087] The invention will be more fully understood by reference to the following examples. However, they should not be construed as limiting the scope of the invention.

[0088] The following examples are offered for illustrative purposes only and are not intended in any way to limit the scope of the present invention.

EXAMPLES

[0089] Commercially available reagents referred to in the examples were used according to the manufacturer's instructions unless otherwise indicated. The source of the cells identified in the following examples, and throughout memory, by ATCC access numbers is the American Type Culture Collection, Manassas, Virginia.

EXAMPLE 1

Expression of DR4 ECD as immunoadhesin

[0090] An immunoadhesin construct with DR4 ECD was prepared. A mature DR4 ECD sequence (amino acids 1-218 shown in Figure 1) was cloned into a Flag vector pCMV-1 (Kodak) in the 3 'direction of the Flag flag sequence and fused to the CH1, hinge and Fc region of the heavy chain of the human immunoglobulin G1 as described previously [Aruffo et al., Cell, 61: 1303-1313 (1990)]. Immunoadhesin was expressed by transient transfection in human 293 cells and purified from cell supernatants by protein A affinity chromatography, as described by Ashkenazi et al., Supra.

EXAMPLE 2

Preparation of specific monoclonal antibodies for DR4

[0091] Balb / c mice (obtained from Charles River Laboratories) were immunized by injecting 0.5 µg / 50 µl of an immunoadhesin protein with DR4 ECD (described in example 1 above) (diluted in MPL adjuvant -RDM purchased from Ribi Immunochemical Research Inc., Hamilton, MT) 11 times in each plant of the hind legs at intervals of 3-4 days.

[0092] Three days after the final stimulation, popliteal lymph nodes were removed from the mice and a suspension of individual cells was prepared in DMEM medium (obtained from Biowhitakker Corp.) supplemented with 1% penicillin-streptomycin. Next, the lymph node cells were fused with murine myeloma cells P3X63AgU.1 (ATTC CRL 1597) using 35% polyethylene glycol and cultured in 96-well culture plates. The hybridomas resulting from the fusion in HAT medium were selected. Ten after fusion, hybridoma culture supernatants were screened in an ELISA to analyze the presence of monoclonal antibodies that bind to the immunoadhesin protein with DR4 ECD (described in example 1).

[0093] In the ELISA, the 96-well microtiter plates (Maxisorb; Nunc, Kamstrup, Denmark) were coated by the addition of 50 µl of 2 µg / ml of goat anti-human IgG Fc (purchased from Cappel Laboratories) in PBS to each well and incubation at 4 ° C overnight. The plates were then washed three times with wash buffer (PBS containing 0.05% Tween 20). Next, the wells of the microtiter plates were blocked with 50 µl of 2.0% bovine serum albumin in PBS and incubated at room temperature for 1 hour. Then, the plates were washed again three times with wash buffer.

[0094] After the washing step, 50 µl of 0.4 µg / ml of EC4 immunoadhesin protein with DR4 ECD in an assay buffer was added to each well. The plates were incubated for 1 hour at room temperature in a shaker, followed by three washes with wash buffer.

[0095] After the washing steps, 100 µl of the hybridoma supernatants or purified antibody with G-sepharose protein columns (10 µg / ml) were added to the designated wells. 100 µl of conditioned medium with P3X63AgU.1 myeloma cells was added to other wells designated as controls. The plates were incubated at room temperature for 1 hour in a stirring apparatus and then three washes were made with wash buffer.

[0096] Next, 50 µl of HRP-conjugated goat anti-mouse IgG Fc (purchased from Cappel Laboratories), diluted 1: 1000 in assay buffer (0.5% bovine serum albumin, was added to each well , 0.05% Tween-20 in PBS) and the plates were incubated for 1 hour at room temperature in a shaker. The plates were washed three times with wash buffer followed by the addition to each well of 50 µl of substrate (TMB peroxidase substrate, Kirkegaard & Perry, Gaithersburg, MD) and incubation at room temperature for 10 minutes. The reaction was stopped by adding to each well 50 µl of TMB-1 component stop solution (diethylglycol, Kirkegaard & Perry) and absorbance at 450 nm was read in an automated microtiter plate reader.

[0097] Hybridoma supernatants initially screened in the ELISA were considered for their ability to bind DR4-IgG, but not CD4-IgG. The ELISA positive supernatants were subsequently analyzed by FACS analysis using 9D cells (a human lymphoid B cell line expressing DR4; Genentech, Inc.) and goat anti-mouse IgG conjugated to FITC. For this analysis, 25 µl of suspended cells (at 4 x 10 6 cells / ml) in cell sorting buffer (PBS containing 1% FCS and 0.02 NaN3 at 0.02) were added to U-shaped microtiter wells %), were mixed with 100 µl of culture supernatant or purified antibody (10 µg / ml) in cell sorting buffer and incubated for 30 minutes on ice. The cells were then washed and incubated with 100 µl of goat anti-mouse IgG conjugated to FITC for 30 minutes at 4 ° C. The cells were then washed twice, resuspended in 150 µl of cell sorting buffer, and then analyzed by FACScan (Becton Dickinson, Mountain View, CA).

[0098] Figure 2 shows FACS staining of 9D cells. Two particular antibodies 4E7.24.3 and 4H6.17.8, recognized the DR4 receptor in 9D cells.

EXAMPLE 3

Assay of the ability of DR4 antibodies to agonistically induce apoptosis

[0099] Hybridoma supernatants and purified antibodies (as described in Example 2 above) were analyzed for their activity to induce DR4-mediated apoptosis in 9D cells. 9D cells (5 X 105 cells / 0.5 ml) were incubated with 1 µg of DR4 mAbs (4E7.24.3 or 4H6.17.8; see example 2 above) or IgG control antibodies in 200 µl of complete RPMI medium at 4 ° C for 15 minutes. The cells were then incubated for 5 minutes at 37 ° C with or without 10 µg of goat anti-mouse IgG Fc antibody (ICN Pharmaceuticals) in 300 µl of complete RPMI. At this point, the cells were incubated overnight at 37 ° C and in the presence of 7% CO2. Next, the cells were collected and washed once with PBS. The viability of the cells was determined by staining the binding of FITC-annexin V to phosphatidylserine according to the manufacturer's recommendations (Clontech). The cells were washed in PBS and resuspended in 200 µl of binding buffer. 10 µl of annexin-V-FITC (1 µg / ml) and 10 µl of propidium iodide were added to the cells. After incubation for 15 minutes in the dark, 9D cells were analyzed by FACS.

[0100] As shown in Figure 3, both DR4 antibodies (in the absence of goat anti-mouse IgG Fc) induced apoptosis in 9D cells compared to control antibodies. However, the agonistic activity of both DR4 antibodies increased by cross-linking the DR4 receptor in the presence of goat anti-mouse IgG Fc (see Figure 4). This greater apoptosis (Figure 4) by both DR4 antibodies is comparable with the apoptotic activity of Apo-2L in 9D cells (data not shown).

EXAMPLE 4

Assay of the ability of the DR4 antibody to block apoptosis of Apo-2L-induced 9D

[0101] Hybridoma supernatants and purified antibodies (as described in Example 2 above) were analyzed for their activity to block apoptosis induced by Apo-2 ligand in 9D cells. 9D cells (5 X 105 cells / 0.5 ml) were suspended in complete RPMI medium (RPMI plus 10% FCS, glutamine, nonessential amino acids, penicillin, streptomycin, sodium pyruvate) and placed in individual Falcon 2052 tubes. 0.5 ml of Apo-2L (1 µg / ml; Apo-2L labeled with soluble His prepared as described in WO 97/25428) was suspended in complete RPMI medium, pre-incubated with DR4 antibody (4H6.17.8 ) serially diluted and / or an Apo-2 antibody (mAb 3F11, Genentech, Inc.), and then added to the tubes containing 9D cells. The 9D cells were incubated on ice for 15 min and then incubated overnight at 37 ° C and in the presence of 7% C02. Then, the incubated cells were collected and washed once with PBS. The viability of the cells was determined by staining the binding of FITCanexin V to phosphatidylserine according to the manufacturer's recommendations (Clontech). Specifically, the cells were washed in PBS and resuspended in 200 µl of binding buffer. 10 µl of annexin-V-FITC (1 µg / ml) and 10 µl of propidium iodide were added to the cells. After incubation for 15 minutes in the dark, 9D cells were analyzed by FACS.

[0102] The results are shown in Figure 5. Since 9D cells express more than one Apo2L receptor, Apo-2L can induce apoptosis in 9D cells by interacting with DR4 or the receptor referred to as Apo- 2. Thus, to detect any blocking activity of DR4 antibodies, it was necessary to block the interaction between Apo-2 and Apo-2L. In combination with the anti-Apo-2 antibody, 3F11, the DR4 antibody 4H6.17.8 was able to block approximately 50% of Apo-2L-induced apoptosis. The remaining apoptotic activity of approximately 50% is believed to be due to the agonistic activity of DR4 antibodies alone, as shown in Figure 5. Accordingly, it is believed that the 4H6.17.8 antibody is an antibody of DR4 lock.

EXAMPLE 5

Antibody isotyping

[0103] The isotypes of antibodies 4H6.17.8 and 4E7.24.3 (as described above) were determined by covering microtiter plates with isotype-specific goat anti-mouse Ig (Fisher Biotech, Pittsburgh, PA) overnight at 4 ° C. Next, the plates were washed with wash buffer (as described in example 2 above). Then, the wells of the microtiter plates were blocked with 200 µl of 2% bovine serum albumin and incubated at room temperature for 1 hour. The plates were washed again three times with wash buffer.

[0104] Next, 100 µl of 5 µg / ml of purified DR4 antibodies or 100 µl of the hybridoma culture supernatant was added to the designated wells. Plates were incubated at room temperature for 30 minutes and then 50 µl of HRP-conjugated goat anti-mouse IgG was added to each well (as described above). The plates were incubated for 30 minutes at room temperature. The level of HRP bound to the plate was detected using HRP substrate as described above.

[0105] Isotyping analysis showed that antibodies 4H6.17.8 and 4E7.24.3 are IgG1 antibodies.

EXAMPLE 6

ELISA assay to analyze the binding of DR4 antibodies to other Apo-2L receptors

[0106] An ELISA was performed to determine if the two DR4 antibodies described in example 2 were capable of binding to other known Apo-2L receptors in addition to DR4. Specifically, DR4 antibodies for Apo-2 binding were analyzed [see, for example, Sheridan et al., Science, 277: 818-821 1 (1997)], DcR1 [Sheridan et al., Supra], and DcR2 [Marsters et al., Curr. Biol., Al., 7: 1003-1006 (1997)]. The ELISA was performed essentially as described in example 2 above.

[0107] The results are shown in Figure 6. The DR4 4E7.24.3 antibody bound to DR4, but not to any of the other Apo-2L, Apo-2, DcR1 or DcR2 receptors. In contrast, the DR4 4H6.17.8 antibody showed some cross reactivity with Apo-2, but not with DcR1 or DcR2.

Material deposit

[0108] The following materials have been deposited with the American Type Culture Collection, 10801 University Boulevard, Manasssas, Virginia, United States (ATCC):

Material ATCC Dep. No. Deposit date 4E7.24.3 HB-12454 January 13, 1998

4H6.17.8 HB12455 January 13, 1998

[0109] This deposit was made as stipulated in the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the purposes of the Patent Procedure and the Regulations under it (Budapest Treaty). This ensures the maintenance of a viable culture of the deposit for 30 years from the date of deposit. The deposit will be available through the ATCC under the terms of the Budapest Treaty, and are subject to an agreement between Genentech, Inc. and ATCC, which ensures the permanent and unrestricted availability of the progeny of the deposit culture for public use after publication. of the respective US patent or after leaving open to the public inspection of any US or foreign patent application, whichever is first, and ensures the availability of the progeny for someone determined by the United States Patent and Trademark Commissioner to have the right thereto in accordance with 35 USC § 122 and the rules of the Commissioner according to them (including 37 CFR § 1.14 with specific reference to 886 OG 638).

[0110] The assignee of the present application has agreed that if a crop of the materials in the deposit died or was lost or destroyed when grown under the proper conditions, the materials will be immediately replaced in a notification by other equals. The availability of deposited material should not be construed as a license to carry out the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws.

[0111] The previously written report is considered to be sufficient to allow a person skilled in the art to carry out the invention. The present invention is not limited in scope by the deposited construction, since the deposited embodiment is intended to be an individual illustration of certain aspects of the present invention and any construction that is functionally equivalent is within the scope of the invention. The deposit of the material of the present invention does not constitute an admission that the written description contained in the present invention is inadequate to allow the practice of any aspect of the invention, including the optimal mode thereof, nor is it construed as limiting the scope from the claims to the specific illustrations it represents. In fact, various modifications of the present invention will be apparent to those skilled in the art from the above description, in addition to those shown and described herein, and are within the scope of the appended claims.

Claims (18)

one.

Isolated agonist monoclonal antibody that (a) specifically binds to a death receptor polypeptide 4 (DR4) comprising amino acid residues 24-218, 1-218, or 1-468 of Figure 1 (SEQ ID NO: 1) , (b) induces apoptosis in at least one type of mammalian cell that expresses the DR4 polypeptide and (c) does not bind to decoy receptors DcR1 or DcR2,

2.

Antibody according to claim 1, which is a human antibody.

3.

Antibody according to claim 1, which is a chimeric antibody.

Four.

Antibody according to claim 1, which is a humanized antibody.

5.

Antibody according to claim 1, wherein said mammalian cell is a cancer cell.

6.

Antibody according to claim 1, wherein the antibody binds to the same epitope as (1) the epitope to which the monoclonal antibody produced by the hybridoma cell line deposited under the American Type Culture Collection of Access Number ATCC HB- binds. 12454 or (2) the epitope to which the monoclonal antibody produced by the hybridoma cell line deposited under the American Type Culture Collection of Access Number ATCC HB-12455 binds.

7.

Hybridoma cell line producing the antibody according to claim 1 or claim 6.

8.

Hybridoma cell line according to claim 7, which has been deposited under the American Type Culture Collection of Access Numbers ATCC HB-12454 or ATCC HB12455.

9.

Monoclonal antibody according to claim 1, produced by the hybridoma cell line according to claim 8.

10.

Isolated nucleic acid encoding the DR4 antibody according to any one of claims 1 to 6.

eleven.

Composition comprising the antibody according to any one of claims 1 to 6 and a carrier.

12.

Composition according to claim 11, wherein said carrier is a pharmaceutically acceptable carrier.

13.

In vitro or ex vivo induction method of apoptosis in mammalian cells comprising exposing mammalian cells to an effective amount of an antibody according to any one of claims 1 to 6.

14.

Method according to claim 13, wherein said mammalian cells are cancer cells.

fifteen.

Use of an antibody according to any one of claims 1 to 6 in the manufacture of a medicament for inducing apoptosis in mammalian cells.

16.

Use according to claim 15, wherein said mammalian cells are cancer cells.

17.

Manufacture article, comprising a container and a composition contained in said container, wherein the composition includes an antibody according to any one of claims 1 to 6.

18.

Dimeric molecule comprising an antibody according to any one of claims 1 to 6 bound to a heterologous immunoglobulin.

COUNT COUNT